Atypical PKC (aPKC) activation appears to be required for increases in glucose transport, and, most importantly, this activation is defective in obesity and type 2 diabetes. Thus, it is critical to elucidate mechanisms used by insulin, exercise and other agents to activate aPKCs. Although initial findings suggest that IRS-1 is required for insulin activation of aPKCs, thiazolidinediones (TZDs) were reported to activate aPKCs and glucose transport in 3T3/L1 adipocytes via CbI/PI3K, independently of IRS-1/2. Importantly, insulin activates CbI/PI3K to the same extent as TZDs in 3T3/L1 adipocytes, and, while this is small relative to IRS-1/PI3K, initial findings suggest that CbI/PI3K is required for activation of aPKCs and glucose transport by insulin. Accordingly, IRS-1 and Cbl may be co-required for PI3K and aPKC activation in these and other cells. To examine this possibility (a) Cbl mutants will be expressed in 3T3/L1 adipocytes and L6 myocytes to see if pYXXM motifs in Cbl are required for activation of the SH2 domain of p85/PI3K, aPKCs and glucose transport; and (b) mice and brown adipocytes in which IRS-1 or IRS-2 has been knocked out will be used to see if IRS-1/2 are required for activation of aPKCs and glucose transport in muscle and adipocytes. Concerning exercise, we have reported that exercise and AICAR activate aPKCs, AICAR uses ERK and phospholipase D (PLD) to activate aPKCs, and aPKCs are required for AICAR-stimulated glucose transport. To see if aPKCs are required for exercise-stimulated glucose transport, we will use transgenic mice that express kinase-inactive aPKCs or muscle-specific aPKC knockout mice. The see if AICAR uses PYK2 to sequentially activate ERK, PLD, aPKCs and glucose transport, we will use viral-mediated expression of mutant forms of these signaling factors. Finally, the hypothesis that insulin-stimulated glucose transport is dependent on aPKCs will be definitively tested in adipocytes and myocytes derived from embryonic stem cells in which PKC-lambda has been knocked out by recombinant methods.